Nicotine receptor agonists in stem cell and progenitor cell recruitment

ABSTRACT

The present invention features methods for recruitment of bone marrow-derived stem cells (e.g., endothelial cell precursors, hematopoietic stem cells) by administration of nicotine or other nicotine receptor agonist. The methods of the invention can be used in, for example, treatment of conditions amenable to treatment by recruitment of bone marrow-derived stem cells (e.g., neutropenia).

CROSS-REFERENCE

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/627,643, filed Jul. 28, 2000, which application claims thebenefit of prior U.S. Provisional Application Ser. No. 60/146,233, filedJul. 28, 1999, and of prior U.S. Provisional Application Ser. No.60/188,300, filed Mar. 10, 1999, which applications are incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates generally to the field of mobilization andrecruitment of stem cells and progenitor cells.

BACKGROUND OF THE INVENTION

Recruitment and renewal of stem cells and/or progenitor cells areimportant in a variety of applications. Prior to bone marrowtransplantation, e.g., expansion and mobilization of the pool ofhematopoietic stem cells is critical. Repopulation of the stem cellcompartment post irradiation or following chemotherapy also requiresexpansion or renewal of hematopoietic stem cells. Other disorders thatwould benefit from proliferation and/or mobilization of stems cellsand/or progenitor cells include blood cell dyscrasias such asneutropenia, leucopenia, acquired immunodeficiencies, and the like.

Vasculogenesis, which involves the growth of vessels derived fromendothelial progenitor cells, is a further example of a process thatinvolves recruitment and/or renewal of stems cells and/or progenitorcells. Vasculogenesis, as well as angiogenesis, the process by which newblood vessels are formed from extant capillaries, and the factors thatregulate these processes, are important in embryonic development,inflammation, and wound healing, and also contribute to pathologicconditions such as tumor growth, diabetic retinopathy, rheumatoidarthritis, and chronic inflammatory diseases (see, e.g., U.S. Pat. No.5,318,957; Yancopoulos et al. (1998) Cell 93:661-4; Folkman et al.(1996) Cell 87;1153-5; and Hanahan et al. (1996) Cell 86:353-64).

Both angiogenesis and vasculogenesis involve the proliferation ofendothelial cells. Endothelial cells line the walls of blood vessels;capillaries are comprised almost entirely of endothelial cells. Theangiogenic process involves not only increased endothelial cellproliferation, but also comprises a cascade of additional events,including protease secretion by endothelial cells, degradation of thebasement membrane, migration through the surrounding matrix,proliferation, alignment, differentiation into tube-like structures, andsynthesis of a new basement membrane. Vasculogenesis involvesrecruitment and differentiation of mesenchymal cells into angioblasts,which then differentiate into endothelial cells which then form de novovessels (see, e.g., Folkman et al. (1996) Cell 87:1153-5).

Several angiogenic and/or vasculogenic agents with different propertiesand mechanisms of action are well known in the art. For example, acidicand basic fibroblast growth factor (FGF), transforming growth factoralpha (TGF-α) and beta (TGF-β), tumor necrosis factor (TNF),platelet-derived growth factor (PDGF), vascular endothelial cell growthfactor (VEGF), and angiogenin are potent and well-characterizedangiogenesis-promoting agents. In addition, both nitric oxide andprostaglandin (a prostacyclin agonist) have been shown to be mediatorsof various angiogenic growth factors, such as VEGF and bFGF. However,the therapeutic applicability of some of these compounds, especially assystemic agents, is limited by their potent pleiotropic effects onvarious cell types.

Angiogenesis and vasculogenesis have been the focus of intense interestsince these processes can be exploited to therapeutic advantage.Stimulation of angiogenesis and/or vasculogenesis can aid in the healingof wounds, the vascularizing of skin grafts, and the enhancement ofcollateral circulation where there has been vascular occlusion orstenosis (e.g., to develop a “biobypass” around an obstruction due tocoronary, carotid, or peripheral arterial occlusion disease). Inaddition, identification of agents that can stimulate recruitment ofstem cells and/or progenitor cells could be useful in the treatment ofother conditions associated with cellular injury and/or depletion ofcells (e.g., acquired or genetic immune deficiencies). There is anintense interest in factors such agents that are well-tolerated by thesubject, but that are of high potency in effecting stimulation of stemcell and/or progenitor cell recruitment.

Literature

Villablanca ((1998) “Nicotine stimulates DNA synthesis and proliferationin vascular endothelial cells in vitro,” J. Appl. Physiol. 84:2089-98)studied the effects of nicotine on endothelial DNA synthesis, DNArepair, proliferation, and cytoxicity using cultures of bovine pulmonaryartery endothelial cells in vitro. The reference Carty et al. ((1996)“Nicotine and cotinine stimulate secretion of basic fibroblast growthfactor and affect expression of matrix metalloproteinases in culturedhuman smooth muscle cells,” J Vasc Surg 24:927-35) demonstrate thatnicotine stimulates vascular smooth muscle cells to produce fibroblastgrowth factor, and also upregulates the expression of several matrixmetalloproteinases. The investigators propose that these datademonstrate mechanisms by which smoking may cause atherosclerosis andaneurysms.

The reference by Belluardo et al. ((1998) Acute intermittent nicotinetreatment produces regional increases of basic fibroblast growth factormessenger RNA and protein in the tel-and diencephalon of the rat,”Neuroscience 83:723-40) reported that nicotine stimulates the expressionof fibroblast growth factor-2 in rat brain, which the investigatorspropose may explain the neuroprotective effect of nicotine in the ratbrain.

Moffett et al. ((1998) “Increased tyrosine phosphorylation and novelcis-actin element mediate activation of the fibroblast growth factor-2(FGF-2) gene by nicotinic acetylcholine receptor. New mechanism fortrans-synaptic regulation of cellular development and plasticity,” MolBrain Res 55:293-305) report that nicotine stimulates the expression offibroblast growth factor-2 in neural crest-derived adrenalpheochromatocytes utilizing a unique transcriptional pathway thatrequires tyrosine phosphorylation. The authors propose that thesefindings suggest that activation of nicotine receptors may be involvedin neural development.

Cucina et al. ((1999) “Nicotine regulates basic fibroblastic growthfactor and transforming growth factor β₁ production in endothelialcells,” Biochem Biophys Res Commun 257:302-12) report that nicotineincreases the release of bFGF, decreases the release of TGFβ1 fromendothelial cells, and increases endothelial mitogenesis. The authorsconclude that these effects may have a key role in the development andprogression of atherosclerosis.

Volm et al. (1999) “Angiogenesis and cigarette smoking in squamous celllung carcinomas: an immunohistochemical study of 28 cases.” AnticancerRes 19(1A):333-6 reports that angiogenesis in lung tumors is linked to apatient's smoking habits.

Macklin et al. (1998) “Human vascular endothelial cells expressfunctional nicotinic acetylcholine receptors,” J. Pharmacol. Exper.Therap. 287:435-9 reports that endothelial cells express both functionalnicotinic (neuronal type) and muscarinic acetylcholine receptors.

U.S. Pat. Nos. 5,318,957; 5,866,561; and 5,869,037 describe use ofvarious compounds (haptoglobin and estrogen) and methods(adenoviral-mediated gene therapy of adipocytes) to effect angiogenesis.

Heeschen et al. ((2001) “Nicotine stimulates angiogenesis and promotestumor growth and atherosclerosis.” Nat Med July; 7(7):833-9) reportsthat nicotine induces angiogenesis, and increases capillary andcollateral growth in vivo.

Jacobi et al. ((2001) “Nicotine stimulates wound healing in diabeticmice.” Am J Pathol July; 161(1):97-104) reports that nicotineaccelerates wound healing in diabetic mice by promoting angiogenesis.

Heeschen et al. ((2003) “Nicotine promotes arteriogenesis.” J Am CollCardiol, February 5; 41(3):489-96) reports that nicotine promotesangiogenesis and arteriogenesis in the setting of ischemia.

Heeschen et al. ((2002) “A novel angiogenic pathway mediated bynon-neuronal nicotinic acetylcholine receptors.” J Clin Invest August;110(4):527-36) reports that pharmacological inhibition of nAChRinhibited inflammatory angiogenesis and reduced ischemia-inducedangiogenesis and tumor growth.

Zhu et al. ((2003) “Second Hand Smoke Stimulates Tumor Angiogenesis andGrowth.” Cancer Cell 4(3):191-6) reports that tobacco smoke promotestumor angiogenesis and growth.

For recent reviews in the field of angiogenesis and vasculogenesis, see,e.g.,

Yancopoulos et al. (1998) Cell 93:661-4; Folkman et al. (1996) Cell87;1153-5; and Hanahan et al. (1996) Cell 86:353-64.

For recent reviews in the field of hematopoiesis, see, e.g., Kondo etal. (2003) Ann. Rev. Immunol. 21:759-806; Prohaska et al. (2002) Semin.Immunol. 14:377-384; Kondo eta 1. (2001) Curr. Opin. Genet. Dev.11:520-526; and Weissman et al. (2001) Ann. Rev. Cell Dev. Biol.17:387-403.

SUMMARY OF THE INVENTION

The present invention features methods for recruiting/mobilizing and/orincreasing proliferation of bone marrow-derived stem cells (e.g.,hematopoietic stem cells) and progenitor cells (e.g. endothelial cellprecursors), as well as methods for recruiting/mobilizing and/orincreasing proliferation of the progeny of such cells, by administrationof nicotine or other nicotine receptor agonist. The methods of theinvention can be used in, for example, treatment of conditions amenableto treatment by recruitment of bone marrow-derived stem cells (e.g.,neutropenia, leukopenia, etc.).

One object of the present invention to provide a method of recruitingendothelial progenitor cells to enhance angiogenesis.

Another object of the present invention is to provide a method oftreating and preventing diseases and ailments involving tissue damage(e.g., to facilitate cellular repair), such as in myocardial andcerebral infarctions, heart failure, mesenteric or limb ischemia,wounds, and vascular occlusion or stenosis.

Another object of this invention is to provide a method of acceleratingwound healing, vascularization and incorporation of skin grafts,musculocutaneous flaps or other surgically transplanted tissues; or toenhance the healing of a surgically created anastomosis.

Another object of the invention is provide a method of treatingconditions or diseases (e.g., blood dyscrasias, therapeutic radiationtreatments, and the like) associated with depletion of cells thatdevelop from bone marrow-derived stem cells (e.g., immune cells, e.g.,neutrophils, eosinophils, T cells, B cells, macrophages, natural killercells, and the like).

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the methods of the invention and compositions used therein asmore fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the chemical structures for the nicotine receptoragonists nicotine, epibatidine, and ABT-154, and for the nicotinereceptor antagonists hexamethonium and mecamylamine.

FIG. 2 is a graph illustrating the effect of oral nicotine uponfibrovascular growth in an animal model.

FIG. 3 is a graph illustrating the effect of locally administerednicotine upon fibrovascular growth in an animal model using the discangiogenesis system.

FIG. 4 is a graph illustrating comparing the relative angiogenicpotencies of the angiogenic factors Del-1 and bFGF with locally orsystemically administered nicotine.

FIG. 5 is a graph illustrating the relative capillary densities innon-ischemic (control; “non-ischemic”) ischemic limbs in the hind limbischemia model (“ischemic”).

FIG. 6 is a graph illustrating the effect of intramuscularlyadministered nicotine upon capillary density in an animal model ofischemia.

FIG. 7 is a graph illustrating the effect of intramuscularlyadministered nicotine upon capillary density in an untreated,non-ischemic limb of an animal of FIG. 6.

FIG. 8 is a schematic illustrating an experimental model of mouseparabiosis used in assays for tracking migration of circulating cellssuch as progenitor cells.

FIG. 9 is a graph showing the capillary density (capillaries/myocyte)and the percentage of new vessels incorporating endothelial progenitorcells for saline control animals and nicotine treated animals.

FIG. 10 is a graph depicting the effect of nicotine (oral administrationfor 6 weeks) on peripheral white blood cell count (WBC).

FIG. 11 is a graph depicting the effect of nicotine on total cellularpopulation of bone marrow (BM) and spleen after 6 weeks ofadministration.

FIG. 12 is a graph depicting the effect of nicotine on the pool ofhematopoietic stem cells (kit/sca⁺) in the bone marrow.

FIGS. 13A and 13B are graphs depicting the effect of a 6-week exposureon long term, self-renewing hematopoietic stem cells (LTHSC; FIG. 13A)and short term, differentiating hematopoietic stem cells (STHSC; FIG.13B) in the bone marrow of C57B16 mice.

DEFINITIONS

The term “nicotine receptor agonist” is meant to encompass nicotine(which is understood to include nicotine derivatives and like compounds)and other compounds that substantially specifically bind a nicotinereceptor and provide a pharmacological effect, e.g., recruitment of stemcells to a treatment site, as in induction of angiogenesis. “Nicotinereceptor agonists” encompass naturally-occurring compounds (including,but not limited to, small molecules, polypeptides, peptides, etc.,particularly naturally-occurring plant alkaloids, and the like),endogenous ligands (e.g., purified from a natural source, recombinantlyproduced, or synthetic, and further including derivatives and variantsof such endogenous ligands), and synthetically produced compounds (e.g.,small molecules, peptides, etc.).

The term “nicotine” is intended to mean the naturally occurring alkaloidknown as nicotine, having the chemical nameS-3-(1-methyl-2-pyrrolidinyl)pyridine, which may be isolated andpurified from nature or synthetically produced in any manner. This termis also intended to encompass the commonly occurring salts containingpharmacologically acceptable anions, such as hydrochloride,hydrobromide, hydroiodide, nitrate, sulfate or bisulfate, phosphate oracid phosphate, acetate, lactate, citrate or acid citrate, tartrate orbitartrate, succinate, maleate, fumarate, gluconate, saccharate,benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorate and pamoate salts. Nicotine is a colorless to paleyellow, strongly alkaline, oily, volatile, hygroscopic liquid having amolecular weight of 162.23 and the formula shown in FIG. 1.

Unless specifically indicated otherwise, the term “nicotine” furtherincludes any pharmacologically acceptable derivative or metabolite ofnicotine which exhibits pharmacotherapeutic properties similar tonicotine. Such derivatives, metabolites, and derivatives of metabolitesare known in the art, and include, but are not necessarily limited to,cotinine, norcotinine, nornicotine, nicotine N-oxide, cotinine N-oxide,3-hydroxycotinine and 5-hydroxycotinine or pharmaceutically acceptablesalts thereof. A number of useful derivatives of nicotine are disclosedwithin the Physician's Desk Reference (most recent edition) as well asHarrison's Principles of Internal Medicine.

The term “nicotine receptor” as in “nicotine receptor agonist” is meantto encompass the classic pentameric protein of the nicotine receptor(formed by subunits which are symmetrically arranged around a centralion channel) as well as any protein comprising a nicotine binding sitethat stimulates recruitment of stem cells or progenitor cells (e.g., asin angiogenesis) upon binding to nicotine or other nicotine receptoragonist (e.g., the muscarinic acetylcholine receptor). Use of the term“nicotine receptor” in the phrase “nicotine receptor agonist” is notmeant to limit the present invention to a theorized mechanism throughwhich nicotine or other nicotine receptor agonists stimulatestem/progenitor cell recruitment (e.g., by binding a nicotine receptor),but rather is a means of describing the types of compounds contemplatedby the invention that can be used to facilitate stimulation ofstem/progenitor cell recruitment.

The terms “long-term” and “short-term,” as used herein to refer tohematopoietic stem cells (HSCs), are art-recognized terms. In general,long-term HSCs (LTHSCs)are self-renewing stem cells. In general,short-term HSCs (STHSCs) are programmed to differentiate intohematopoietic progenitor cells. Human LTHSCs can be distinguished fromSTHSCs using methods that are known in the art. For example, humanLTHSCs are generally CD38⁻, while human STHSCs are generally CD38⁺. Inmouse, LTHSCs are generally Flk-2⁻, while STHSCs are generally Flk-2⁺.See, e.g., Christiansen and Weissman ((2001) Proc. Natl. Acad. Sci. USA98:14541-14546; Chen et al. ((2002) Proc. Natl. Acad. Sci. USA99:15468-15473; Hogan et L. ((2002) Proc. Natl. Acad. Sci. USA99:413-418; deWynter et al. ((1998) Stem Cells 16:387-396;

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect, e.g., stimulation of angiogenesis and/or vasculogenesis. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment” as used herein covers anytreatment of a disease in a mammal, particularly a human, and includes:(a) preventing a disease or condition (e.g., preventing the loss of askin graft or a re-attached limb due to inadequate vascularization) fromoccurring in a subject who may be predisposed to the disease but has notyet been diagnosed as having it; (b) inhibiting the disease, e.g.,arresting its development; or (c) relieving the disease (e.g., enhancingthe development of a “bio-bypass” around an obstructed vessel to improveblood flow to an organ). For example, in the context of the presentinvention, stimulation of angiogenesis and/or vasculogenesis is employedfor subject having a disease or condition amenable to treatment byincreasing vascularity and increasing blood flow.

By “therapeutically effective amount of a nicotine receptor agonist” ismeant an amount of a nicotine receptor agonist effective to facilitate adesired therapeutic effect, e.g., a desired level of angiogenic and/orvasculogenic stimulation. The precise desired therapeutic effect willvary according to the condition to be treated.

By “isolated” is meant that the compound is separated from all or someof the components that accompany it in nature.

By “substantially pure nicotine receptor agonist” is meant that thenicotine receptor agonist has been separated from components thataccompany it in nature. Typically, a nicotine receptor agonist issubstantially pure when it is at least 50% to 60%, by weight, free fromnaturally-occurring organic molecules with which it is naturallyassociated. Generally, the preparation is at least 75%, more preferablyat least 90%, and most preferably at least 99%, by weight, nicotinereceptor agonist. A substantially pure nicotine receptor agonist can beobtained, for example, by extraction from a natural source (e.g.,tobacco), by chemically synthesizing the compound, or by a combinationof purification and chemical modification. A substantially pure nicotinereceptor agonist can also be obtained by, for example, enriching asample having nicotine receptor agonist activity for a factor or factorsthat provide such activity, e.g., by obtaining a fraction havingincreased nicotine receptor agonist activity. Purity can be measured byany appropriate method, e.g., chromatography, mass spectroscopy, HPLCanalysis, etc.

For example, a nicotine receptor agonist is substantially free ofnaturally associated components when it is separated from thosecontaminants which accompany it in its natural state. Thus, a nicotinereceptor agonist which is chemically synthesized or produced in acellular system different from the cell from which it naturallyoriginates will be substantially free from its naturally associatedcomponents

The term “stem cell” is used herein to refer to a mammalian cell thathas the ability both to self-renew, and to generate differentiatedprogeny (see Morrison et al. (1997) Cell 88:287-298). Generally, stemcells also have one or more of the following properties: an ability toundergo asynchronous, or symmetric replication, that is where the twodaughter cells after division can have different phenotypes; extensiveself-renewal capacity; capacity for existence in a mitotically quiescentform; and clonal regeneration of all the tissue in which they exist, forexample the ability of hematopoietic stem cells to reconstitute allhematopoietic lineages. “Progenitor cells” differ from stem cells inthat they typically do not have the extensive self-renewal capacity, andoften can only regenerate a subset of the lineages in the tissue fromwhich they derive, for example only lymphoid, or erythroid lineages in ahematopoietic setting. The term “stem/progenitor cell” used throughoutis used for simplicity, is not meant to be limiting, and is meant toencompass stem cells, progenitor cells, and both stem and progenitorcells.

Stem cells may be characterized by both the presence of markersassociated with specific epitopes identified by antibodies and theabsence of certain markers as identified by the lack of binding ofspecific antibodies. Stem cells may also be identified by functionalassays both in vitro and in vivo, particularly assays relating to theability of stem cells to give rise to multiple differentiated progeny.“Stem cells” is meant to include, but is not necessarily limited to,hematopoietic stem cells and progenitor cells derived therefrom (U.S.Pat. No. 5,061,620); neural crest stem cells (see Morrison et al. (1999)Cell 96:737-749); embryonic stem cells; mesenchymal stem; mesodermalstem cells; etc.

Other hematopoietic “progenitor” cells of interest include cellsdedicated to lymphoid lineages, e.g. immature T cell and B cellpopulations. The methods of the present invention are useful inexpanding selected populations of these cells.

“Mesenchymal progenitor cells” is meant to include, but is notnecessarily limited to, endothelial progenitor cells and other cellsdedicated to development into cells of mesenchymal lineages, e.g.,connective tissue, cartilage, chondrocytes, bone (osteoblasts), fatcells (adipocytes), and the outer layers of blood vessels.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anicotine receptor agonist” includes a plurality of such agonists andreference to “the nicotine receptor” includes reference to one or morenicotine receptors and equivalents thereof known to those skilled in theart, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

Overview of the Invention

The present invention is based on the surprising discovery that nicotineinduces recruitment of endothelial progenitor cells to blood vessels andprovides for induction of angiogenesis. The inventors have also foundthat peripheral white blood cell count is increased by nicotine, aneffect that is associated with an increase in the numbers of short-termand long-term hematopoietic cells in the bone marrow.

The inventors' initial inquiries were based on the clinical observationthat smokers often have inadequate collateral development after coronaryor peripheral arterial obstruction, i.e., the inventors at firstsuspected that nicotine might play a role in inhibition of angiogenesis.Accordingly, the inventors began by testing the local effects ofnicotine in the disc angiogenesis system (DAS). Unexpectedly, theinventors discovered that nicotine was as or more potent as anangiogenic agent than any growth factor tested in this system, includingDel1 (Penta et al. (1999) J Biol Chem 274(16):11101-9) and bFGF.Additional studies revealed that the potent angiogenic effects ofnicotine were mediated in part by products of the cyclooxygenasecascade, and in part by the NO synthase pathway. Studies using the discangiogenesis system suggested that nicotine may be useful fortherapeutic angiogenesis. However, because angiogenesis is such acomplex process, to demonstrate proof of principle that an agent hasutility for therapeutic angiogenesis, the agent was tested in an animalmodel of disease that requires angiogenesis for its treatment.Accordingly, studies were performed in an animal model of arterialocclusion (the murine hind limb ischemia model). Using this model ofarterial occlusion, the inventors obtained compelling evidence thatnicotine induces therapeutic angiogenesis.

In addition, the inventors discovered that induction of angiogenesis bynicotine involves mobilization and recruitment of endothelial progenitorcells to the site of injury. This discovery suggests that nicotine caninduce mobilization and recruitment of stem cells and progenitor cells,and thus can be used to treat conditions in which a cell population hasbeen depleted due to, for example, infection, physical damage,autoimmunity, etc.

Thus, the inventors have discovered that nicotine, a component oftobacco smoke, provides the basis of a new therapeutic approach toenhance angiogenesis in the treatment of coronary, peripheral, or otherocclusive arterial diseases; for the enhancement of wound healing andthe improved vascularization of surgically transplanted tissues ororgans(e.g., skin grafts or reattached limbs); and for the recruitmentand mobilization of stem cells and progenitor cells to provide forrepopulation of a depleted or damaged mature cell population.

In view of its similar or relatively enhanced potency relative to other,conventional angiogenic agents, nicotine has significant advantage overcurrent candidates as the basis of therapeutic angiogenesis. Moreover,the pharmacology and pharmacokinetics of nicotine have already beenwell-characterized in the context of smoking (e.g., in an effort tofacilitate smoking cessation) and methods for slow release and localdelivery have already been intensively investigated. Processes for themanufacture of nicotine and nicotine agonists are also wellcharacterized. Furthermore, these small molecules are more easilysynthesized and stored than complex angiogenic peptides.

Accordingly, the invention encompasses methods and compositions formobilization and recruitment of stem cells and/or progenitor cells, byadministration of a nicotine receptor agonist.

Nicotine and Other Nicotine Receptor Agonists

The methods of the invention are accomplished by administration of anicotine receptor agonist, particularly nicotine, nicotine metabolite,or nicotine derivative. Methods for production of nicotine derivativesand analogues are well known in the art. See, e.g., U.S. Pat. No.4,590,278; 4,321,387; 4,452,984; 4,442,292; and 4,332,945.

Additional nicotine receptor agonists of interest include, but are notnecessarily limited to, naturally occurring plant alkaloids (e.g.,lobeline, lobeline derivatives, and the like), which plant-derivedcompounds can be provided in a herbal preparation (e.g., in the form ofdried tobacco leaves, in a poultice, in a botanical preparation, etc.),in isolated form (e.g., separated or partially separated from thematerials that naturally accompany it), or in a substantially purifiedform. Other nicotine receptor agonists include choline esteraseinhibitors (e.g., that increase local concentration of acetylcholine),derivatives of epibatidine that specifically bind the neuronal type ofnicotinic receptors (with reduced binding to the muscarinic receptor)and having reduced deleterious side-effects (e.g., Epidoxidine, ABT-154,ABT-418, ABT-594; Abbott Laboratories (Damaj et al. (1998) J. PharmacolExp. Ther. 284:1058-65, describing several analogs of epibatidine ofequal potency but with high specificity to the neuronal type ofnicotinic receptors). Further nicotine receptor agonists of interestinclude, but are not necessarily limited to, N-methylcarbamyl andN-methylthi-O-carbamyl esters of choline (e.g., trimethylaminoethanol)(Abood et al. (1988) Pharmacol. Biochem. Behav. 30:403-8); acetylcholine(an endogenous ligand for the nicotine receptor); and the like.

Nicotine receptor agonists can also be readily identified using methodswell known in the art. For example, the ability of a candidate nicotinereceptor agonist can be screened for binding to a nicotine receptor invitro, and the ability of the candidate agent to recruit stem cellsand/or progenitor cells can be assessed in vivo (e.g., using the discangiogenesis system (DAS), in the hind limb ischemia model, etc.).

Pharmaceutical Compositions

Upon reading the present specification, the ordinarily skilled artisanwill appreciate that the pharmaceutical compositions comprising anicotine receptor agonist described herein can be provided in a widevariety of formulations. More particularly, the nicotine receptoragonist can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid(e.g., gel), liquid or gaseous forms, such as tablets, capsules,powders, granules, ointments, solutions, suppositories, injections,inhalants and aerosols. Where the nicotine receptor agonist is anaturally-occurring compound, the pharmaceutical composition can also beprovided as an herbal preparation (e.g., in the form of tobacco leaves,as a poultice of plant matter, in a botanical preparation, etc.).

The nicotine receptor agonist formulation used will vary according tothe condition or disease to be treated, the route of administration, theamount of nicotine receptor agonist to be administered, and othervariables that will be readily appreciated by the ordinarily skilledartisan. In general, and as discussed in more detail below,administration of nicotine receptor agonists can be either systemic orlocal, and can be achieved in various ways, including, but notnecessarily limited to, administration by a route that is enteral (e.g.,oral), parenteral, intravenous, intravascular, intra-arterial,inter-pericardial, intramuscular, intraperitoneal, transdermal,transcutaneous, subdermal, intradermal, intrapulmonary, etc.

In pharmaceutical dosage forms, the nicotine receptor agonist may beadministered in the form of their pharmaceutically acceptable salts, orthey may also be used alone or in appropriate association, as well as incombination, with other pharmaceutically active compounds. The followingmethods and excipients are merely exemplary and are in no way limiting.

The nicotine receptor agonist can be formulated into preparations forinjection by dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

Formulations suitable for topical, transcutaneous, and transdermaladministration, e.g., to administer the nicotine receptor agonistdirectly to a wound, may be similarly prepared through use ofappropriate suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. Topical formulations may be alsoutilized with a means to provide continuous administration of nicotineor other nicotine receptor agonist by, for example, incorporation intoslow-release pellets or controlled-release patches.

The nicotine receptor agonist can also be formulated in a biocompatiblegel, which gel can be applied topically (e.g., to facilitate woundhealing) or implanted (e.g., to provide for sustained release ofnicotine receptor agonist at an internal treatment site). Suitable gelsand methods for formulating a desired compound for delivery using thegel are well known in the art (see, e.g., U.S. Pat. Nos. 5,801,033;5,827,937; 5,700,848; and MATRIGEL™).

For oral preparations, the nicotine receptor agonist can be used aloneor in combination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The nicotine receptor agonist can be utilized in aerosol formulation tobe administered via inhalation. The compounds of the present inventioncan be formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the nicotine receptor agonist can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. The compounds of the present invention canbe administered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and/or animalsubjects, each unit containing a predetermined quantity of nicotinereceptor agonist calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the unit dosageforms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

In addition to one or more nicotine receptor agonists, thepharmaceutical formulations according to the invention can comprise orbe administered in parallel with additional active agents. For example,where the pharmaceutical formulation is to be administered to promoteangiogenesis and/or vasculogenesis, the formulation can compriseadditional agents to further enhance angiogenesis by enhancing nitricoxide (NO) levels (e.g., by enhancing activity of NO synthase, byenhancing release of NO, etc.) or prostacyclin levels (e.g., byenhancing activity prostacyclin synthase, by enhancing release ofprostacyclin, etc.) Exemplary NO level-enhancing agents include, but arenot necessarily limited to, L-arginine, L-lysine, and peptides enrichedwith these amino acids which can serve as substrates for NO; agents thatpreserve NO activity such as antioxidants (e.g., tocopherol, ascorbicacid, ubiquinone) or antioxidant enzymes (e.g., superoxide dismutase);and agents which can enhance NO synthase activity (e.g.,tetrahydrobiopterin, or precursors for tetrahydrobiopterin (e.g.,sepiapterin)); and the like. Exemplary prostacyclin level-enhancingagents include, but are not limited to precursors for prostacyclin suchas eicosopentanoic acid and docosohexanoic acid; and prostanoids such asprostaglandin El and its analogues; and the like. Alternatively or inaddition, the pharmaceutical compositions according to the invention cancomprise additional agents for stimulation of stem cell and/orendothelial cells recruitment that act through pathways other than thenicotine receptor (e.g., in angiogenesis or vasculogenesis, VEGF, FGF(e.g., aFGF, bFGF), Del-1, etc.).

Particularly where the nicotine receptor agonist is to be delivered forlocal application, e.g., by an intramuscular route, it may be desirableto provide the nicotine receptor agonist in a gel or matrix. The gel ormatrix can, for example, provide at least the initial substrate uponwhich, for example, new tissue can form. For example, the gel or matrixcan be extruded into an ischemic region to form a path for new bloodvessel formation so as to bypass an obstruction in the area.

Administration of Nicotine Receptor Agonists forMobilization/Recruitment of Stem and Progenitor Cells In Vivo

In order to accomplish mobilization or recruitment of stem cells and/orprogenitor cells, nicotine or other nicotine receptor agonists can beadministered in any suitable manner, preferably with pharmaceuticallyacceptable carriers. One skilled in the art will readily appreciate thatthere are available a variety of suitable methods of administeringnicotine or other nicotine receptor agonist in the context of thepresent invention , and, although more than one route can be used toadminister a particular compound, a particular route can provide a moreimmediate, more effective, and/or associated with fewer side effectsthan another route. In general, a nicotine receptor agonist can beadministered according to the method of the invention by, for example,an enteral (e.g., an oral), a parenteral, intravenous, intra-arterial,inter-pericardial, intramuscular, intraperitoneal, transdermal,transcutaneous, subdermal, intradermal, or intrapulmonary route.

The instant methods provide for stimulating proliferation of stem cellsand/or progenitor cells, or the progeny of such cells. The instantmethods provide for stimulating mobilization of stem cells and/orprogenitor cells, or the progeny of such cells, from the bone marrow toa treatment site in a mammal. Treatment sites include, but are notlimited to, the bloodstream, the peripheral circulation, lymph nodes, awound or ulcer, and the like. In some embodiments, the methods providefor incorporation of circulating stem cells or progeny thereof into thetreatment site.

In order to avoid the side-effects associated with systemic nicotine, itmay be preferable to administer nicotine locally (either alone or withadditional active agents, e.g., to enhance the activity of the NOsynthase or prostacyclin synthase pathways in stimulation ofangiogenesis; or to facilitate development of stem or progenitor cellsinto the desired mature cells (e.g., endothelial cells). Localadministration can be accomplished by, for example, direct injection(e.g., intramuscular injection) at the desired treatment site, byintroduction of the nicotine receptor agonist formulation intravenouslyat a site near a desired treatment site (e.g., into a vessel orcapillary that feeds a treatment site), by intra-arterial orintra-pericardial introduction, by introduction (e.g., by injection orother method of implantation) of a nicotine receptor agonist formulationin a biocompatible gel or capsule within or adjacent a treatment site,by injection directly into muscle or other tissue in which increasedblood flow and/or increased vascularity is desired and/or to whichrecruitment of stem cells or progenitor cells is desired, by rectalintroduction of the formulation (e.g., in the form of a suppository to,for example, facilitate vascularization of a surgically createdanastomosis after resection of a piece of the bowel), etc.

In one particular application of interest, the nicotine receptor agonistformulation is employed in a “biobypass” method, wherein instead ofperforming a more invasive procedure, such as a coronary bypassoperation, a nicotine receptor agonist formulation is administered toinduce growth of new blood vessels around the blocked region. In thisembodiment, the nicotine receptor agonist formulation can beadministered in the area of and/or proximate to the ischemic tissue tostimulate angiogenesis.

In some embodiments it may be desirable to deliver the nicotine receptoragonist directly to the wall of a vessel. One exemplary method of vesselwall administration involves the use of a drug delivery catheter,particularly a drug delivery catheter comprising an inflatable balloonthat can facilitate delivery to a vessel wall. Thus, in one embodimentthe method of the invention comprises delivery of a nicotine receptoragonist to a vessel wall by inflating a balloon catheter, wherein theballoon comprises a nicotine receptor agonist formulation covering asubstantial portion of the balloon. The nicotine receptor agonistformulation is held in place against the vessel wall, promotingadsorption through the vessel wall. In one example, the catheter is aperfusion balloon catheter, which allows perfusion of blood through thecatheter while holding the nicotine receptor agonist against the vesselwalls for longer adsorption times. Examples of catheters suitable fornicotine receptor agonist application include drug delivery cathetersdisclosed in U.S. Pat. Nos. 5,558,642; 5,554,119; 5,591,129; and thelike.

In another embodiment of interest, the nicotine receptor agonistformulation is delivered in the form of a biocompatible gel, which canbe implanted (e.g., by injection into or adjacent a treatment site, byextrusion into or adjacent a tissue to be treated, etc.). Gelformulations comprising a nicotine receptor agonist can be designed tofacilitate local release of the nicotine receptor agonist and otheractive agents for a sustained period (e.g., over a period of hours,days, weeks, etc.). The gel can be injected into or near a treatmentsite, e.g., using a needle or other delivery device. In one embodiment,the gel is placed into or on an instrument which is inserted into thetissue and then slowly withdrawn to leave a track of gel, resulting instimulation of stem cell and/or progenitor cell recruitment along thepath made by the instrument. This latter method of delivery may beparticularly desirable for, for example, directing course of abiobypass.

In other embodiments it may be desirable to deliver the nicotinereceptor agonist formulation topically, e.g., for localized delivery,e.g., to facilitate wound healing. Topical application can beaccomplished by use of a biocompatible gel, which may be provided in theform of a patch, or by use of a cream, foam, and the like. Several gels,patches, creams, foams, and the like appropriate for application towounds can be modified for delivery of nicotine receptor agonistformulations according to the invention (see, e.g., U.S. Pat. Nos.5,853,749; 5,844,013; 5,804,213; 5,770,229; and the like). In general,topical administration is accomplished using a carrier such as ahydrophilic colloid or other material that provides a moist environment.Alternatively, for the purpose of wound healing the nicotine agonistcould be supplied, with or without other agents in a gel or cream thecould be applied to the wound. An example of such an application wouldbe as a sodium carboxymethylcellulose-based topical gel with a lowbioburden containing the nicotine agonist and other active ingredientstogether with preservatives and stabilizers.

In other embodiments, the nicotine receptor agonist formulation isdelivered locally or systemically, using a transdermal patch. Severaltransdermal patches are well known in the art for systemic delivery ofnicotine to facilitate smoking cessation, and such patches may bemodified to provide for delivery of an amount of nicotine receptoragonist effective to stimulate stem cell and/or progenitor cellrecruitment according to the invention (see, e.g., U.S. Pat. Nos.4,920,989; and 4,943,435, NICOTROL™ patch, and the like).

In other methods of delivery, the nicotine receptor agonist can beadministered using iontophoretic techniques. Methods and compositionsfor use in iontophoresis are well known in the art (see, e.g., U.S. Pat.Nos. 5,415,629; 5,899,876; 5,807,306; and the like).

The desirable extent of stem/progenitor cell mobilization/recruitmentwill depend on the particular condition or disease being treated, aswell as the stability of the patient and possible side-effects. Inproper doses and with suitable administration, the present inventionprovides for a wide range of development of blood vessels (e.g., fromlittle development to essentially full development), as well as for wideranges of stem cell or progenitor cell mobilization and/or recruitment(e.g., mobilization of a sufficient number of cells to provide forcomplete repopulation of a cell type (e.g., to provide for replenishmentof immune cells in a host immunocompromised following infection,radiotherapy, chemotherapy, and the like; or to provide for localizedrecruitment of stem cells or progenitor cells to a site of local injury(e.g., as in wound healing, local ischemia, etc.)).

The activity of nicotine receptor agonists in stem/progenitor cellmobilization/recruitment can be controlled by administration ofcompounds that interfere with nicotine receptor agonist-mediatedrecruitment. In this sense, the invention also provides for a means ofcontrolling or inhibiting activity of nicotine receptor agonists byinterfering with its role in these processes. This may be accomplished,for example, administration of agents that inhibit the ability of thenicotine receptor agonist to mediate its effects through the nicotinereceptor (e.g., by inhibiting binding to the nicotine receptor).Exemplary nicotinic receptor antagonists include hexamethonium andmecamylamine (formulas provided in FIG. 1). Alternatively, nicotinereceptor agonist-mediated activity can be controlled or inhibited byadministration of inhibitors of processes downstream of nicotinereceptor signaling. For example, inhibitors of nitric oxide synthaseand/or prostacyclin antagonists can be administered to inhibit activityof a nicotine receptor agonist in stimulating angiogenesis. Theinhibitor may be administered in the same manner and dosages to mammals,such as humans, as described with respect to the nicotine receptoragonist.

Where the subject has had exposure, particularly chronic exposure, to anicotine receptor agonist (e.g., as in a subject who is a smoker and whohas had prior, particularly chronic, systemic exposure to nicotine), thenicotine receptor or other nicotine receptor agonist-binding receptorthat mediates stimulation of stem/progenitor cellmobilization/recruitment may be present at lower levels than in subjectswho have not had previous exposure or chronic exposure to a nicotinereceptor agonist. In such subjects, it thus may be desirable toadminister an initial course of a nicotine receptor antagonist tostimulate an increase in nicotine binding-receptors.

Dose

The dose of nicotine or other nicotine receptor agonist administered toa subject, particularly a human, in the context of the present inventionshould be sufficient to effect a desired therapeutic response (e.g.,mobilization/recruitment of stem/progenitor cells in a therapeuticallyeffective number, therapeutic angiogenic response, etc.) in the subjectover a reasonable time frame. The dose will be determined by the potencyof the particular nicotine receptor agonist employed and the conditionof the subject, as well as the body weight of the subject to be treated.For example, the level or affinity or both of the nicotine receptoragonist for the nicotine receptor may play a role in regulating thecompound's activity. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects that mightaccompany the administration of a particular compound.

In determining the effective amount of nicotine or nicotine receptoragonist in the stimulation of stem/progenitor cell recruitment, theroute of administration, the kinetics of the release system (e.g., pill,gel or other matrix), and the potency of the nicotine agonist isconsidered so as to achieve the desired effect with minimal adverse sideeffects. The nicotine receptor agonist will typically be administered tothe subject being treated for a time period ranging from a day to a fewweeks, consistent with the clinical condition of the treated subject.

The following dosages assume that nicotine is being administered, or anicotine receptor agonist with similar potency and efficacy as nicotine.As will be readily apparent to the ordinarily skilled artisan, thedosage is adjusted for nicotine receptor agonists according to theirpotency and/or efficacy relative to nicotine. If given orally or as aninhalant, the dose may be in the range of about 0.01 mg to 10 mg, given1 to 20 times daily, and can be up to a total daily dose of about 0.1 mgto 100 mg. If applied topically, for the purpose of a systemic effect,the patch or cream would be designed to provide for systemic delivery ofa dose in the range of about 0.01 mg to 10 mg. If the purpose of thetopical formulation (e.g., cream) is to provide a local effect, the dosewould likely be in the range of about 0.001 mg to 1 mg. If injected forthe purpose of a systemic effect, the matrix in which the nicotineagonist is administered is designed to provide for a systemic deliveryof a dose in the range of about 0.001 mg to 1 mg. If injected for thepurpose of a local effect, the matrix is designed to release locally anamount of nicotine agonist in the range of about 0.003 mg to 1 mg.

Regardless of the route of administration, the dose of nicotine receptoragonist can be administered over any appropriate time period, e.g., overthe course of 1 to 24 hours, over one to several days, etc. Furthermore,multiple doses can be administered over a selected time period. Asuitable dose can be administered in suitable subdoses per day,particularly in a prophylactic regimen. The precise treatment level willbe dependent upon the response of the subject being treated. In thetreatment of some individuals with nicotine receptor agonists, it may bedesirable to utilize a “megadosing” regimen. In such a treatment, alarge dose of the nicotine receptor agonist is administered to anindividual, time is allowed for the compound to act, and then a suitablereagent, e.g., a nicotine receptor antagonist, is administered to theindividual to render the active compound ineffective or to reduce itssystemic side-effects.

Conditions Amenable to Treatment by Nicotine Receptor Agonist-mediatedAngiogenesis

The methods and nicotine receptor agonist-comprising compositions of theinvention can be used to treat a variety of conditions that wouldbenefit from recruitment of stem/progenitor cells.

In one embodiment, nicotine receptor agonists are used to stimulaterecruitment of endothelial progenitor cells to provide for stimulationof angiogenesis, stimulation of vasculogenesis, increased blood flow,and/or increased vascularity. Use of nicotine receptor agonists in thisembodiment are referred to generally herein as “therapeuticangiogenesis.”

Examples of conditions and diseases amenable to therapeutic angiogenesisaccording to the method of the invention include any conditionassociated with an obstruction of a blood vessel, e.g., obstruction ofan artery, vein, or of a capillary system. Specific examples of suchconditions or disease include, but are not necessarily limited to,coronary occlusive disease, carotid occlusive disease, arterialocclusive disease, peripheral arterial disease, atherosclerosis,myointimal hyperplasia (e.g., due to vascular surgery or balloonangioplasty or vascular stenting), thromboangiitis obliterans,thrombotic disorders, vasculitis, and the like. Examples of conditionsor diseases that can be prevented using the methods of the inventioninclude, but are not necessarily limited to, heart attack (myocardialinfarction) or other vascular death, stroke, death or loss of limbsassociated with decreased blood flow, and the like.

Other forms of therapeutic angiogenesis include, but are not necessarilylimited to, the use of nicotine receptor agonists to accelerate healingof wounds or ulcers; to improve the vascularization of skin grafts orreattached limbs so as to preserve their function and viability; toimprove the healing of surgical anastomoses (e.g., as in re-connectingportions of the bowel after gastrointestinal surgery); and to improvethe growth of skin or hair.

Conditions Amenable to Treatment by Nicotine Receptor Agonist-mediatedInduction of Stem Cell and/or Progenitor Cell Mobilization and/orRecruitment

In addition to stimulation of endothelial progenitor cells inangiogenesis and/or vasculogenesis, nicotine or other nicotine receptoragonists can also be used to induce mobilization and/or recruitmentand/or proliferation (e.g., renewal) of stem cells and/or progenitorcells to provide for replacement of a depleted mature cell or tosupplement a population of mature cells. By “mobilization” is meant theinduction of movement of stem and/or progenitor cells from their site oforigin (e.g., the bone marrow) and into the systemic circulation.“Recruitment” refers to the movement of stem cells and/or progenitorcells from the systemic circulation and to a local site, e.g., to awound or other site of physical damage, site of infection, an organ,etc. In addition to or as part of the mobilization/recruitmentprocesses, nicotine receptor agonists can also induce proliferation andmaturation of stem cells and progenitor cells. Thus, a method ofmobilization or recruitment of stem and/or progenitor cells may involveincreasing the numbers of such cells.

Conditions amenable to treatment by mobilization of stem cells and/orprogenitor cells include, but are not necessarily limited to, conditionsamenable to treatment by increasing the number of immune cells (e.g.,neutrophils, eosinophils, T cells, B cells, macrophages, natural killercells), cells of mesenchymal origin (e.g., cells of connective tissue,cells of cartilage, chondrocytes, bone (osteoblasts), fat cells(adipocytes), and cells of blood vessels, e.g., endothelial cells).Thus, in some embodiments, the invention provides for treating disordersassociated with a reduction in immune cells. Disorders associated with areduction in immune cells include, but are not limited to, neutropenia,leukopenia, an acquired immunodeficiency, and the like. Acquiredimmunodeficiency can result from any of a variety of conditions ortreatments, including, but not limited to, radiation therapy,chemotherapy (e.g., cancer chemotherapy), a viral infection, and thelike.

In some embodiments, the invention provides methods for stimulatingproliferation of bone marrow cells (including, e.g., hematopoietic stemcells, immune progenitor cells, and the progeny of such cells) toprovide for an increased number of such cells in a mammalian subjectprior to obtaining cells from the subject for use in bone marrowtransplantation. For example, an effective amount of a nicotine receptoragonist is administered to an individual who is a prospective bonemarrow donor at a suitable time period before bone marrow is taken fromthe donor for transplantation into a recipient. For example, aneffective amount of a nicotine receptor agonist is administered to aprospective donor at a time period of from about 30 minutes to about 2weeks, e.g., from about 30 minutes to about 1 hour, from about 1 hour toabout 2 hours, from about 2 hours to about 4 hours, from about 4 hoursto about 8 hours, from about 8 hours to about 12 hours, from about 12hours to about 16 hours, from about 16 hours to about 24 hours, fromabout 24 hours to about 2 days, from about 2 days to about 4 days, fromabout 4 days to about 7 days, or from about 1 week to about 2 weeks,prior to obtaining bone marrow from the donor individual.

In some embodiments, the invention provides for stimulation ofproliferation of stem and/or progenitor cells, where the stem and/orprogenitor cells are of hematopoietic lineage. For example, in someembodiments, administration of a nicotine receptor agonist induces an atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, or at least about 50%, or greater,increase in peripheral white blood cell count. In other embodiments,administration of a nicotine receptor agonist induces an at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, or at least about 50%, or greater, increase in totalcell count in the bone marrow. In other embodiments, administration of anicotine receptor agonist induces an at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, or at least about 50%, or greater, increase in short-termhematopoietic stem cells in the bone marrow. In other embodiments,administration of a nicotine receptor agonist induces an at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, or at least about 50%, or greater, increase inlong-term hematopoietic stem cells in the bone marrow.

In one embodiment, the mobilized/recruited cells are mesenchymal stemcells. Mesenchymal stem cells are a population of progenitor cells inthe bone marrow that are capable of differentiating into bone,cartilage, muscle, tendon, and other connective tissues (Bruder et al.(1998) Clin Orthop (355 Suppl):S247-56). Human mesenchymal stem cellsare positive for the markers SH2, SH3 and SH4.

In another embodiment, the mobilized/recruited cells are hematopoieticstem cells. Hematopoietic cells encompass HSCs, erythrocytes,neutrophils, monocytes, platelets, mast cells, eosinophils andbasophils, B and T lymphocytes and NK cells as well as the respectivelineage progenitor cells. As used herein, a hematopoietic stem cell(HSC) refers to a primitive or pluripotential hematopoietic stem cellthat is capable of giving rise to progeny in all defined hematolymphoidlineages: limiting numbers of stem cells are capable of fullyreconstituting lethally irradiated mice, leading to their long-termsurvival. In humans, the CD34⁺ Thy-1⁺ Lin⁻ hematopoietic stem cells arethe equivalent of the murine c-kit⁺ Thy-1.1^(lo) Lin^(+/lo) Sca-1⁺(KTLS) hematopoietic stem cells and are a virtually pure population ofmultilineage hematopoietic stem cells. Human hematopoietic stem cellspresent on their surfaces the markers CD34, thy-1, SCA-1 and SCA-2; andare negative for lineage specific markers which may include glycophorinA, CD3, CD24, CD16, CD14, CD38, CD45RA, CD36, CD2, CD19, CD56, CD66a,and CD66b; T cell specific markers, tumor specific markers, etc. Markersuseful for identification of mesodermal stem cells include FcγRII,FcγRIII, Thy-1, CD44, VLA-4α, LFA-1β, HSA, ICAM-1, CD45, Aa4.1, Sca-1,etc. See also U.S. Pat. Nos. 5,035,994; 5,061,620; 5,061,620; andTerstappen et al. (1992) Blood 79:666-677).

In another embodiment, the mobilized/recruited stem cells are neuralcrest stem cells. Neural crest stem cells are positive for low-affinitynerve growth factor receptor (LNGFR), and negative for the markerssulfatide, glial fibrillary acidic protein (GFAP), myelin protein P_(o),peripherin and neurofilament.

In an embodiment of particular interest, the cells mobilized andrecruited cells are endothelial progenitor cells.

Exemplary conditions amenable to treatment by mobilization/recruitmentof stem/progenitor cells include, but are not necessarily limited toischemia, immune deficiency disorders (e.g., neutropenia, leukopenia,acquired immunodeficiencies, etc.), and hemophilia (e.g., acquiredhemophilia). The methods of the invention can also be used in thetreatment of damage that results from injury (e.g., mechanical,physical, chemical, radiation, nuclear, autoimmune, pathogen-mediatedinjury, and the like).

Conditions amenable to treatment by mobilization/recruitment of stemcells and/or progenitor cells can result from any of a variety ofcauses. For example, reduction of a mature cell population can be adirect or secondary effect of a disease or injury, can result fromtreatment of a disease or injury (e.g., as in chemotherapy for treatmentof cancer). For example, neutropenia, a condition associated withdepletion of neutrophils, can result from bone marrow damage fromcertain types of leukemia, lymphoma or metastatic cancer; adversereactions to medication such as a diuretics or antibiotics; therapiesthat can cause depletion of immune cells, e.g., radiation treatment orchemotherapy; viral infections cause by, for example, EBV virus (e.g.,infectious mononucleosis), HIV, and the like; bacterial infections(e.g., tuberculosis); or autoimmune diseases (e.g., systemic lupuserythromatosis).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric. Standard abbreviations may beused, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s,second(s); min, minute(s); hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); and the like.

Materials and Methods

The following is a description of the methods and materials used in thespecific examples below.

Animals

Eight to ten week old female wild type C57BL/6J mice were used. The miceweighed 20-25 grams (Jackson Laboratories, Bar Harbor, Me. andDepartment of Comparative Medicine (DCM), Stanford, Calif.), and weremaintained as previously described (Maxwell et al. (1998) Circulation1998 98(4):369-374).

Disc Angiogenesis System (DAS)

To study whether nicotine induces angiogenesis in vivo, we employed thedisc angiogenesis system (DAS) (Kowalski et al. (1992) Exp Mol Pathol56(1):1-19; Fajardo et al. (1998) Lab Invest 58:718-7244).

Preparation of the disc The DAS consisted of a disc (11 mm in diameterand 1 mm thickness) made of a polyvinyl alcohol sponge (Kanebo PVA,Rippey Co., Santa Clara, Calif.). Both sides were covered withnitrocellulose cell-impermeable filters (Millipore filters, 0.45 μm inpore diameter, Millipore, SF, Calif.) of the same diameter as the spongedisc, fixed to the sponge using Millipore glue #1 (xx70000.00,Millipore). As a result, cells(and thus vessels) could penetrate or exitonly through the rim of the disc (Kowalski et al. (1992) Exp Mol Pathol56(1):1-19; Fajardo et al. (1998) Lab Invest 58:718-7244).

In order to study the local effect of nicotine on angiogenesis, nicotinewas placed in a pellet which was added directly to the disc. Briefly, a1.5-mm core (pellet) was cut from the disc center. Both the pellets anddiscs were sterilized prior to assembly in a laminar flow hood. Thepellet was loaded with up to 20 mcl (μl) of the nicotine solution andsubsequently air-dried. We placed in the disc pellets with eithervehicle (PBS, Sigma, Chemical Co., St Louis, Mo., n=5) or nicotine (10-6M, Aldrich Chemical Company, Milwaukee, Wis., n=5) to study the effectsof locally administered nicotine. For comparison, in some cases basicfibroblast growth factor(bFGF; 20 mcg) or Del-1 protein(0.2 M) was addedto the pellet, rather than nicotine. Both bFGF and Del-1 are known toinduce angiogenesis. The pellet was then coated with ethylene-vinylacetate co-polymer (Elvax, Dupont, Chemcentral Corp., Chicago, Ill.)which would permit slow release of the nicotine from the pellet into thedisc. The pellet was then re-inserted into the disc before sealing thedisc with the millipore filters.

To study the systemic effects of nicotine, in some cases the animalswere administered nicotine in their drinking water (see below).

Implantation of the disc The mice were anesthetized with 4%chloralhydrate [intraperitoneal administration (i.p.), 0.1 cc/10 g bodyweight]. The flanks and posterior surface of the thorax were shaved andcleaned with saturated 70% isopropyl alcohol. A 2-cm incision was madein the skin of the flank contra-lateral to the implantation site. Bluntdissection through the subcutaneous tissue produced a channel into whichthe saline moistened disc was inserted. The skin was closed with 5.0silk suture (Kowalski et al. (1992) Exp Mol Pathol 56(1):1-19; Fajardoet al. (1998) Lab Invest 58:718-7244).

Disc removal and preparation Two weeks after disc implantation, the micewere sacrificed with an overdose of 4% chloral hydrate (i.p.) andcervical dislocation. A careful incision was made next to the skinoverlying the implanted disc, and the disc was gently removed from theimplantation site. Attached tissue was carefully detached from the disc.After removing the disc, one filter was separated from the disc. Discswere then fixed in 10% formalin and embedded flat in paraffin.Subsequently, 5 μm sections were made in a plane through the center ofthe disc and parallel to the disc surface.

Quantitation of results The disc sections were stained with H&E forlight microscopy and histomorphometric measurement of radial growth, andstained with toluidine blue for quantitative determination of total areaof fibrovascular growth. Using a video microscope and a computerassisted digital image analysis system (NIH Image 1.59b9), the entirearea of fibrovascular growth in the toluidine blue stained disc wascalculated and expressed in mm². As described in a previous study, totalfibrovascular growth area is directly proportional to the total area ofthe disc occupied by blood vessels (Kowalski et al. (1992) Exp MolPathol 56(1):1-19; Fajardo et al. (1998) Lab Invest 58:718-7244).Therefore, the measurement of such total area is used as an index ofangiogenesis (Kowalski et al. (1992), supra; Fajardo et al. (1998),supra).

Vascular continuity assessment To visualize the microvessels in the discsections and to establish that there was continuity between the systemicand disc vasculatures, luconyl blue dye was injected into the leftcarotid artery prior to euthanizing the mice. Animals were anesthetizedusing 4% chloralhydrate (i.p., 0.1 cc/10 g body weight). An incision wasmade in the ventral midline of the neck. After the carotid sheath wasexposed, the left carotid artery was separated from the neurovascularbundle and secured by two 4.0 silk sutures. An incision was made in thecarotid and a 15-cm length of PE10 tubing (Beckton Dickinson, SparksMd.) was introduced into the carotid artery and advanced to theascending aorta just distal to the aortic valve. About 1.0 ml of luconylblue was then slowly injected from a 1 ml syringe through the tubinginto the thoracic aorta. The presence of blue dye in the fibrovascularnetwork in the disc was detected by light microscopy. Microscopyrevealed microvessels lined by a single layer of endothelium anderythrocytes contained within their lumen. Luconyl blue dye was observedthroughout the vessels of the disc.

Murine Ischemic Hind Limb Model of Peripheral Arterial Disease (PAD)

Mice were anesthetized with 4% chloral hydrate [intraperitonealadministration (i.p.), 0.1 cc/10 g body weight). The medial surface ofboth hind limbs were shaved and then cleaned with betadine solution. A1.5-cm longitudinal incision was performed, extending from the knee tothe inguinal ligament. Through this incision, the superficial femoralartery was dissected free along its length. After the distal ends ofboth the external iliac and superficial femoral arteries were ligatedwith 7.0 (Ethicon), complete excision of the femoral artery wasperformed. An additional set of mice underwent sham operation. Theincisions were then closed with discontinuous stitches of 5.0 silksuture (Ethicon). Ampicillin (1 mg/10 gm body weight) intraperitonealinjection was administered after surgical procedure.

Histological Studies

Tissue Preparation. Three weeks after surgery, mice were euthanized withan overdose of 4% chloral hydrate (i.p) and cervical dislocation, andthe adductor and semimembranous muscles were collected for capillarydensity assessment. Briefly, a longitudinal incision in the medial thighwas made to expose the entire hindlimb muscle. The adductor andsemimembranous muscles were removed and immediately frozen in OCT.Subsequently, sections 5 μm were taken from the mid-region of eachmuscle in a transverse orientation. The sections were air dried andfixed in acetone.

Capillary Densitometry. Immunohistochemistry was performed using analkaline phosphatase assay to identify the endothelial cells. An eosincounterstain was used to differentiate myocytes. Capillaries andmyocytes were identified and counted using light microscopy (20×). Foreach section, four different fields were selected and the total numberof capillaries and myocytes per field determined. These values wereaveraged to provide a determination of capillary density for eachexperimental limb. To ensure that value for capillary density was notoverestimated due to muscle atrophy, or underestimated due tointerstitial edema, capillary density was expressed as a ratio ofcapillaries to myocytes present in the same field.

Data Analysis

All data are given as mean+/−SEM. Statistical significance was testedusing unpaired, two-tailed t-test for comparisons between groups.Statistical significance was accepted for p<0.05.

Example 1 Systemic Effect of Nicotine Upon Angiogenesis In Vivo

To study the effects of nicotine in systemically treated mice, nicotine(60 mcg/ml, n=5) was diluted in the drinking water. The mechanism ofnicotine-induced angiogenesis was studied by giving oral supplementationof indomethacin (20 mcg/ml, Sigma, n=5) and/or LNNA (6 mg/ml, Sigma,n=5) to mice having an implanted DAS both locally treated (nicotineinside the DAS) and systemically treated (nicotine diluted in drinkingwater). Concentrations of nicotine, indomethacin, and LNNA weredetermined in accordance to studies using oral supplementation of theseagents in murine models (Maxwell et al. (1998) Circulation 199898(4):369-374; Fulton et al. (1980) Int J Cancer 26(5):669-73; Rowell etal. (1983) J Pharmacological Methods 9:249-261).

Under basal conditions (untreated water, vehicle-treated disc),fibrovascular growth into the disc occurred. Vessels could be seengrowing into the disc. These vessels were in continuity with thesystemic circulation as manifested by the influx of leuconyl dye intothe disc vasculature, after systemic administration of the dye. The areaof the fibrovascular growth into the disc under basal conditions wassomewhat greater than 10 mm² (FIG. 2). With systemic administration ofnicotine, there was a dramatic increase in fibrovascular growth with anarea of 35 mm² (FIG. 2). The effect of nicotine was blocked by the NOsynthase inhibitor L, nitro-arginine (LNNA) as well as by indomethacin,indicating that synthesis of both nitric oxide and prostacyclin wererequired for the angiogenic effect of nicotine.

Example 2 Local Effect of Nicotine Upon Angiogenesis in Vivo

In order to determine if local administration of L-arginine could beeffective at inducing angiogenesis, in some animals, nicotine was placedwithin a pellet that was inserted into the disc angiogenesis system(described above). When nicotine was placed in the disc (rather thanadministered in the water of the animals as described in Example 1) asimilar effect was observed. The fibrovascular growth under basalconditions (about 10 mm²) was increased to about 20 mm² (FIG. 3). Again,indomethacin or LNNA blocked the effects of nicotine. These studiesindicate that systemic or local administration of nicotine inducesangiogenesis.

Example 3 Comparison of Effects of Nicotine with Other Angiogenic Agents

The effects of nicotine were compared with the angiogenic agents bFGFand

Del1. The comparison with bFGF is particularly important because thisagent is already in clinical trials in humans for therapeuticangiogenesis. In comparison to vehicle, bFGF, Del1, and nicotine eachincreased angiogenesis to the same degree. Systemically administerednicotine enhanced angiogenesis to a much greater degree than locallyadministered bFGF and Del-1 (FIG. 4). Paradoxically, the effect ofsystemic nicotine administration was greater than local nicotineadministration, even though systemic nicotine administration undoubtedlyproduced lower local levels in the disc. This paradox led theinvestigators to consider that the systemic administration of nicotinewas inducing vasculogenesis(recruitment of endothelial precursors fromthe bone marrow) as well as local angiogenesis (see Example 5 below).Intermediate doses of nicotine administered intramuscularly had thegreatest effect. At higher intramuscular doses of nicotine, lessangiogenesis is observed.

Example 4 Induction of Angiogenesis in the Murine Hindlimb IschemiaModel of Peripheral Arterial Disease

To provide more compelling evidence for the therapeutic angiogeniceffects of nicotine, the angiogenic effects of nicotine were examined ina model of arterial occlusive disease, the murine ischemic hindlimb(described above). Daily intramuscular injections of nicotine solutionor vehicle were administered (50 μl) for a period of three weeks. Fivegroups of animals received 0, 3, 30, 300 or 3200 ng/kg of nicotine byintramuscular injection daily (represented in FIGS. 6 and 7 as 1×(0.0811 ng nicotine in 50 μl saline (=0.003 μg/kg), 10× (0.811 ngnicotine in 50 μl saline (=0.03 μg/kg), 100× (8.11 ng nicotine in 50 μlsaline (=0.3 μg/kg), and 1000× (81.1 ng nicotine in 50 μl saline (=3.2μg/kg)). As shown in FIG. 5, 3 weeks after surgery, capillary density(capillaries/myocyte) was increased in operated limbs (ischemic) incomparison to non-operated limbs (non-ischemic) consistent with a basalangiogenic response to ischemia.

As shown in FIGS. 6 and 7, nicotine enhanced the angiogenic response toischemia relative to controls. With vehicle control, 0.35capillaries/myocyte were detected in the ischemic limb. At anintermediate dose of 0.03 μg/kg, nicotine nearly doubled angiogenicresponse (to 0.67 capillaries/myocyte). At the highest dose of nicotine,angiogenesis was not increased; indeed at this dose some toxicity wasobserved, with evidence of interstitial edema and myocyte necrosis. Theangiogenic effect remained local to the site of nicotine injection,since no angiogenic effect was detected in a non-ischemic hindlimbs(FIG. 6). Thus, local intramuscular administration of these doses ofnicotine did not result in a systemic angiogenic effect.

These studies indicate that nicotine enhances angiogenesis in a murinemodel of human peripheral arterial disease, and that nicotine receptoragonists are useful for therapeutic angiogenesis.

Example 5 Nicotine Induces Angiogenesis by Recruitment of EndothelialProgenitor Cells

In order to further examine the role of nicotine in induction ofangiogenesis, the recruitment of endothelial progenitor cells wasstudied. The model of mouse parabiosis, in which cross-circulation isestablished between two individuals (Eichwald, et al. (1963) J NatlCancer Inst 30:783-94; Weissman et al. (1984) Transplantation 37:3-6),is an ideal assay for tracking the migration of circulating cells withendothelial potential to sites of angiogenesis in vivo. Cells arisingfrom one partner can be differentiated from the other by virtue ofstable genetic markers such as sex chromosomes or the presence of areporter transgene such as LacZ (FIG. 8). The goal is to eliminatebiases inherent in models that require pre-selection of a given celltype, and to avoid manipulations (such as total body irradiation)required to overcome immunological or physiological barriers between theputative precursor cells and the experimental hosts. The use of highlyinbred laboratory mice allows unparalleled versatility in the use ofcongenic and transgenic markers to perform lineage-tracking experiments.The parabiotic mouse model is an excellent model for studying thebiology and genetic program of endothelial progenitors, and inestablishing the role of circulating precursors in the development ofperivascular supporting structures.

Parabiotic mouse pairs were created to investigate the mobilization andincorporation of precursors to vessel formation in normal and ischemicconditions using a hind limb ischemia model of angiogenesis (asdescribed above), and the effect of nicotine. The parabiotic partnerswere selected so that they shared all major histocompatibility antigens,and were therefore free of immunological barriers to cell migration andangiogenesis. Assaying for genetic markers unique to one animal of thepair (e.g., the Y chromosome where the pair is made up of a female mouseand a male mouse) provides for unambiguous cell tracking between themice. The model is further optimized by the use of a second geneticmarker to differential the individuals (e.g., a transgene present in onepartner and not the other), thus increasing the sensitivity and theaccuracy of the identification of the origin of single cells.

Ten week old male mice (mouse A: either a C57/B16 ROSA 26 mice (JacksonLaboratory, Bar Harbor, Me.) or a tie-2-LacZ (“Sato”) transgenic mouse(Schlager et al. (1995) Development 121(4):1089-98; Schlager et al.(1997) Proc Natl Acad Sci USA 94(7):3058-63) that has endothelialspecific β-galactosidase expression), which constitutively express theLacZ transgene, were surgically joined to a female age andstrain-matched mice (mouse B: either C57/B6 (LacZ⁻⁻) mouse (to be joinedwith the ROSA26 mouse) or a C57B16 mouse (to be joined with thetie-2-LacZ mouse) by making a continuous unilateral skin incisionbetween the fore and hindlimb joints of each mouse. An anastomosis ofthe leg joints and the skin with its adherent subcutaneous tissue ofmouse A to mouse B was then made with nylon sutures, and the miceallowed to recover. Previous studies using this model have shown thatcross-circulation is reliably established within 2 weeks (Eichwald, etal. (1963) J Natl Cancer Inst 30:783-94; Weissman et al. (1984)Transplantation 37:3-6). Cross-circulation in these animals wasconfirmed by tracking the flow of Evans blue dye from mouse A to mouse Bfollowing intravenous injection. In addition, peripheral bloodleukocytes (pbls) were tested for contributions from each partner byFACS-gal staining (Fiering et al. (1991) Cytometry 12:291-301) orY-chromosome FISH to guarantee that both cells and plasma freelyinterchange. Weissman et al. have shown previously that parabiosis ofsyngeneic males to females does not result in a detectable anti H-Yimmune response (Weissman et al. (1984) Transplantation 37:3-6).

The LacZ⁻ females were exposed to hindlimb ischemia whereas the LacZ⁺males were not. Test mice were treated with nicotine systemically byadding nicotine to the drinking water (0.1 g/L drinking water). Controlmice did not receive nicotine in their drinking water. Five pairs ofparabiotic mice were included in each of the test and control groups.

Ischemic and non-ischemic hindlimbs were removed from the female mice at3 weeks after induction of the ischemia and examined for the presence ofcells derived from their male partners. The phenotype of the cells inthe hindlimbs were evaluated using histology methods by confocalmicroscopy. Double staining for CD 31 and β-galactosidase identifiedEPCs derived from the transgenic mouse. EPC frequency was defined as thenumber of vessels containing transgenic endothelial cells divided by thetotal vessels examined in representative sections.

Results

Examination of ischemic hind limbs from the legs of control andnicotine-treated female partners joined with a Sato/tie-2/LacZtransgenic male showed that EPCs from Sato mouse crossed over to thearea of angiogenesis in the female mouse and incorporated into newvessels. These EPCs were readily identified as they were double stainedfor CD 31 (endothelial marker) and β-galactosidase (which is underendothelial cell-specific tie-2 promoter control in the Sato mice).

The results are illustrated graphically in FIG. 9. The percentage ofvessels that incorporated endothelial progenitor cells (EPCs) duringangiogenesis in response to hind limb ischemia was low in the salinegroup (1.6%). However, stimulation of the native angiogenic response bynicotine resulted in a significant increase in the number of vessels andin the percentage of vessels that incorporated EPC (7.3%; P<0.001).

Example 6 Nicotine Increases Long-Term and Short-term Populations ofHematopoietic Stem Cells

Methods

Mice (C57B16j) were given solution containing vehicle (0.4% saccharinein distilled water) or nicotine (100 μg/ml) ad libitum in their drinkingwater. After 6 weeks, peripheral blood, bone marrow and spleen wereharvested. Cell counts were obtained in the blood, spleen and bonemarrow, and bone marrow cells were isolated for fluorescent activatedcell sorting (FACS). To detect hematopoietic stem cells (HSCs),fluorescently labeled antibodies directed against the following antigenswere used: Lineage antigens(CD3,4,5, and 8; B220;Grl, Mac1 and Ter119),cKit, Sca 1, and Flk2. HSCs were conventionally defined as Lin−, cKit+,Sca1+, and Flk2+. All cells were stained with PI (propidium iodide) toseparate live from dead cells.

Results

At the time of sacrifice, the mice appeared healthy. Nicotine levelswere measured, and found to be in the range observed in the plasma oflight to moderate smokers (87 ng/ml). In the nicotine treated animals,white blood cell count was elevated (FIG. 10). Thus, oral administrationof nicotine increases the peripheral white blood cell count (WBC).

In addition, as shown in FIG. 11, the cellularity of the bone marrow andthe spleen were increased. FIG. 11 presents data showing that oraladministration of nicotine increases bone marrow and spleen cellularity(i.e., the total cellular population of BM and spleen is increased). Asshown in FIG. 12, FACS analysis of the bone marrow cell populationrevealed a significant increase in hematopoietic stem cells (kit/sca⁺cells). This was due to equal increases in the long term HSCs(self-renewing cells), and an increase in the short term HSCs (which aredestined to differentiate into progenitors), as shown in FIGS. 13A and13B, respectively.

The data indicate that nicotine increases hematopoietic stem cells, boththe short and long term subpopulations. Furthermore, this increase inHSCs leads to an increase in the cellularity of the bone marrow andspleen, as well as an increase in the white blood cell count. These dataindicate that nicotine receptor agonists enhance stem cell andprogenitor cell populations, and are therefore useful for treatingdeficiencies related to insufficient amounts of these cells, or injuriesor dysfunctions that may be responsive to stem cell therapy. Thesedeficiencies include a variety of conditions, including blood dyscrasiassuch as neutropenia; or insufficient blood supply as is observed inmyocardial ischemia and heart failure.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method for stimulating proliferation and mobilization of stem cellsor their progeny in a mammal, the method comprising administering to amammal a nicotine receptor agonist in an amount effective to stimulate astem cell or progeny thereof to proliferate and to mobilize from bonemarrow of the mammal to a treatment site in the mammal, wherein thenicotine receptor agonist is selected from nicotine, a nicotine salt, anicotine derivative, a nicotine metabolite, a derivative of a nicotinemetabolite, cotinine, norcotinine, nicotine N-oxide, cotinine N-oxide,3-hydroxycotinine, and 5-hydroxycotinine, and wherein the agonist iseffective to treat a disorder selected from neutropenia, leukopenia, oran acquired immunodeficiency resulting from radiation, chemotherapy, ora viral infection other than human immunodeficiency virus, or whereinthe agonist is effective to increase immune cells in the mammal prior toobtaining cells from the mammal for use in bone marrow transplantation.2. The method of claim 1, wherein the nicotine receptor agonist isnicotine, a nicotine salt, a nicotine derivative, a nicotine metabolite,or a derivative of a nicotine metabolite.
 3. The method of claim 1,wherein said administering is by local administration.
 4. The method ofclaim 1, wherein said administering is by systemic administration. 5.The method of claim 1, wherein said administering is oral.
 6. The methodof claim 1, wherein said administering is parenteral.
 7. The method ofclaim 1, wherein said administering is intramuscular.
 8. The method ofclaim 1, wherein said administering is intravascular.
 9. The method ofclaim 1, wherein the treatment site is in the bloodstream of the mammal.10. The method of claim 1, wherein the treatment site is in theperipheral circulation.
 11. The method of claim 1, wherein the treatmentsite is a wound or ulcer.
 12. The method of claim 1, wherein the stemcell is of hematopoietic or mesenchymal lineage.
 13. The method of claim1, wherein the agonist is effective to stimulate incorporation ofcirculating stem cells or progeny thereof into the treatment site. 14.The method of claim 1, wherein the agonist is effective to promote woundhealing at the treatment site.
 15. The method of claim 1, wherein thedisorder is neutropenia.
 16. The method of claim 1, wherein the disorderis leukopenia.
 17. The method of claim 1, wherein the disorder isacquired immunodeficiency, and wherein the acquired immunodeficiencyresults from radiation therapy.
 18. The method of claim 1, wherein thedisorder is acquired immunodeficiency, and wherein the acquiredimmunodeficiency results from cancer chemotherapy.
 19. The method ofclaim 1, wherein the agonist is effective to increase immune cells inthe mammal prior to obtaining cells from the mammal for use in bonemarrow transplantation.
 20. The method of claim 1, wherein the agonistis effective to increase the number of white blood cells in theperipheral blood.
 21. The method of claim 1, wherein the agonist iseffective to increase the number of long-term hematopoietic stem cellsin the bone marrow.
 22. The method of claim 1, wherein the agonist iseffective to increase the number of short-term hematopoietic stem cellsin the bone marrow.
 23. The method of claim 1, wherein the agonist isselected from cotinine, norcotinine, nicotine N-oxide, cotinine N-oxide,3-hydroxycotinine, and 5-hydroxycotinine.